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Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency

a technology of cobalamin receptor and polypeptide, which is applied in the field of transcobalamin receptor polypeptides and polynucleotides, can solve the problems of inability to accurately report amino acid and carbohydrate analyses, inability to provide information about the source of tc in vivo, and inability to synthesise tc in liver cells in culture, etc., and achieve the effect of reducing the uptake of transcobalamin

Active Publication Date: 2015-06-02
THE RES FOUND OF STATE UNIV OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

These modulators effectively inhibit or enhance cobalamin uptake, allowing for targeted treatment of conditions like cancer and inflammatory disorders by selectively depleting or delivering cobalamin to cells, thereby arresting cellular proliferation or promoting therapeutic delivery of agents to tumor cells.

Problems solved by technology

Any perturbation of this process will ultimately lead to intracellular Cbl deficiency.
In addition, studies showing TC synthesis by liver cells, macrophages, fibroblasts, lymphocytes and ileal mucosa in culture do not provide information about the source of TC in vivo.
Based on the specific activity of the protein reported, and protein content, at best the final product would be 60% pure and therefore, amino acid and carbohydrate analyses reported cannot be accurate.
They did not provide any data on the yield of functional activity, purity or specific activity of the final product.
However, clear evidence of Cbl deficiency in AD and other dementias is lacking.
The metabolic pathways involving these two vitamins have not been adequately characterized in brain tissue.
A role for Cbl and folate deficiency in various dementias could not be established by measuring total Cbl and folate levels in serum and CSF because they have provided ambiguous results.
In the absence of dietary deficiency, decreased cellular uptake may cause intracellular deficiency.
In humans, Cbl deficiency due to malabsorption or poor dietary intake takes several years to present clinically because of the large liver stores of Cbl.
However they develop Cbl deficiency rapidly, i.e., after several months following birth because they lack TC.
It has been difficult to produce an animal model of Cbl deficiency by Cbl depletion because of hepatic stores and contribution to the Cbl pool by the gut microflora.
It has been even more difficult to produce Cbl deficiency in vitro in cultured cells because of the TC-Cbl contribution from serum or serum factor supplements required for in vitro cultures.
However, a carefully controlled study has shown that all cells have an absolute requirement for Cbl, and fail to replicate when intracellular Cbl decreases below a critical concentration.

Method used

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  • Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency
  • Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency
  • Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency

Examples

Experimental program
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Effect test

example 1

Purification and Characterization of the Transcobalamin Receptor

[0311]This example describes the purification and characterization of the TCblR protein.

Purification of TCblR

[0312]The initial protocol for the preparation of placental membranes, solubilization of TCblR and affinity purification was based on the original procedure of Seligman and Allen except for the use of aminopropyl-Cbl coupled to Sephacryl or Emphaze matrix on which rabbit TC was immobilized. This purification yielded a product that was only 25% pure based on functional activity and multiple protein bands were identified by LC-MS in the region corresponding to the expected size of TCblR when separated by SDS-PAGE and stained with Coomassie blue.

[0313]A facile functional assay to monitor the TC-Cbl binding activity of TCblR proved very useful to follow the progress during purification. This assay utilized either wheat germ agglutinin (WGA) or concanavalin A (Con A)-agarose to bind TCblR. This method takes advantage ...

example 2

Identification and Characterization of the Transcobalamin Receptor cDNA and Gene

[0326]A search of the human genome data bank revealed the complete gene sequence and chromosomal localization of the human TCblR gene (FIG. 5). The cDNA sequence of human TCblR is provided in SEQ ID NO:2.

Identification of Cis Genomic Elements that Regulate TCblR Expression

[0327]Since TCblR expression is regulated with maximum expression in actively dividing cells, this differential expression is used to characterize transcriptional regulation of this gene and to identify trans-active factors involved in this regulation. Studies on the level of TCblR transcript and protein expression in various primary cell lines such as peripheral skin fibroblasts, venous endothelial cells and in malignant cells such as K562, HL 60 and ECV 304 provide information on the precise time frame for high- or low-TCblR expression and TCblR expression throughout the cell cycle.

[0328]The regulation of TCblR expression is analyzed ...

example 3

Characterization of Transcobalamin Receptor Expression in Human Tissues

[0343]The availability of gene probes and antiserum to TCblR facilitate direct identification of the TCblR mRNA transcript and protein in tissue sections, respectively, by in situ hybridization and immunohistochemistry.

[0344]The techniques for both procedures are well established. Polyclonal antiserum specific for TCblR was obtained from R&D Systems (goat anti-human 8D6 polyclonal antibody; Minneapolis, Minn.). The polyclonal antiserum to TCblR reacts well in immunohistochemical analysis as shown in FIG. 18. NIH IMAGE software and a phosphor-imager were used to quantify the autoradiographic signal, or the fluorescence signal intensity in tissue sections.

[0345]Tissues for these analyses are obtained from commercial sources for tissue arrays because these have been collected under controlled conditions and tested for integrity of proteins and mRNA using specific markers. The utility of tissue microarrays in normal ...

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Abstract

The present invention provides the amino acid and polynucleotide sequences of the transcobalamin receptor, as well as modulators of the transcobalamin receptor. Accordingly, the present invention provides compositions and methods for the treatment and prevention of diseases and disorders associated with cobalamin deficiency, including compositions and methods that promote cobalamin uptake. In addition, the present invention provides compositions and methods for the detection, treatment, and prevention of diseases associated with deregulated cell growth, including, e.g., cancer and autoimmune disorders, including compositions and methods that inhibit cobalamin uptake.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 12 / 296,254 filed on Aug. 24, 2009, which is a national stage application of International Application No. PCT / US2007 / 008674, filed on Apr. 5, 2007, which claims priority to U.S. Provisional Patent Application Ser. No. 60 / 790,330, filed on Apr. 7, 2006, each of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT INTEREST[0002]This invention was made in part with funds provided by the United States Government. Accordingly, the United States Government may have certain rights to this invention.STATEMENT REGARDING SEQUENCE LISTING[0003]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is KYIP—020—02US_ST25.txt. The text file is 14 KB, was created on Aug. 13, 2013, and is...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/28G01N33/53C12P21/08G01N33/567A61K39/395
CPCA61K39/39583C07K16/18C07K16/2896C07K2317/76C07K14/705
Inventor QUADROS, EDWARD V.SEQUEIRA, JEFFREY M.
Owner THE RES FOUND OF STATE UNIV OF NEW YORK
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